Drifting two-dimensional separation with adaption of second dimension gradient to actual first dimension condition

ABSTRACT

A control device for a sample separation apparatus, the sample separation apparatus including a first separation unit and a second separation unit downstream of the first separation unit and supplied with the fluidic sample after treatment by the first separation unit. A control device is configured for controlling the first separation unit to execute a primary separation sequence within a time interval for separating the fluidic sample into fractions, and for controlling the second separation unit to execute secondary separation sequences within the time interval for further separating the separated fractions into sub-fractions, wherein the secondary separation sequences form part of a common sample separation method defined by a common specification of the sample separation involving a set of parameters, and adjusting, over a progress of the primary separation sequence, at least one parameter according to which at least one of the plurality of secondary separation sequences is executed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application under 37 C.F.R.§1.53(b) of commonly owned U.S. patent application Ser. No. 13/179,138,filed on Jul. 8, 2011 and naming Klaus Witt, et al. as inventors.Priority is claimed under 35 U.S.C. §120 from U.S. patent applicationSer. No. 13/179,138, and the entire disclosure of U.S. patentapplication Ser. No. 13/179,138 is specifically incorporated herein byreference.

The present invention relates to a sample separation system.

BACKGROUND

In liquid chromatography, a fluidic sample and an eluent (liquid mobilephase) may be pumped through conduits and a column in which separationof sample components takes place. The column may comprise a materialwhich is capable of separating different components of the fluidicanalyte. Such a packing material, so-called beads which may comprisesilica gel, may be filled into a column tube which may be connected toother elements (like a control unit, containers including sample and/orbuffers) by conduits. The composition of the mobile phase can beadjusted by composing the mobile phase from different fluidic componentswith variable contributions.

Two-dimensional separation of a fluidic sample denotes a separationtechnique in which a first separation procedure in a first separationunit is performed to separate a fluidic sample into a plurality offractions, and in which a subsequent second separation procedure in asecond separation unit is performed to further separate the plurality offractions into sub-fractions. Two-dimensional liquid chromatography (2DLC) may combine two liquid chromatography separation techniques.

Pavel Jandera, Tomas Hajek, Petr Cesla, “Comparison of varioussecond-dimension gradient types in comprehensive two-dimensional liquidchromatography”, J. Sep. Sci. 2010, 33, 1382-1397, discloses thatgradient elution provides significant improvement in peak capacity withrespect to isocratic conditions. In the second dimension, gradients arelimited to a short-time period available for separation. Various typesof second-dimension gradients in comprehensive LC LC are compared: (i)“full in fraction”, (ii) “segment in fraction” and (iii) “continuouslyshifting” gradients, applied in orthogonal LC LC separations of phenolicacids and flavones on a polyethylene glycol column in the firstdimension and two types of porous shell fused-core C18 columns in thesecond dimension (Ascentis Express and Kinetex). The porous shellcolumns provide narrow bandwidths and fast second-dimension separationsat moderate operating pressure that allows important savings of theoverall separation time in comprehensive LC LC separations. The effectsof the gradient type on the bandwidths, theoretical peak capacity,separation time and column pressure in the second dimension wereinvestigated. The type of gradient program controls the range oflipophilicity of sample compounds that can be separated in thesecond-dimension reversed-phase time period. This range can becalibrated using alkylbenzene standards, to design the separationconditions for complete sample separation, avoiding harmful wrap aroundof non-eluted compounds to the subsequent second-dimension fractions.

However, such a concept of two-dimensional liquid chromatography iscumbersome for a user since the second dimension is divided into aplurality of completely unrelated gradient runs, involving the need fora user to program each of these gradient runs individually.

Furthermore, when plotting the result of a 2D LC measurement in atwo-dimensional coordinate system, it may happen that relatively largearea regions may remain empty or basically empty. This corresponds tothe fact that a certain portion of the measurement time is spentinefficiently.

SUMMARY

It is an object of the invention to provide an efficiently operatingtwo-dimensional sample separation apparatus. The object is solved by theindependent claims. Further embodiments are shown by the dependentclaims.

According to an exemplary embodiment of the present invention, a controldevice for a sample separation apparatus for separating a fluidic sampleis provided, the sample separation apparatus comprising a firstseparation unit supplyable with the fluidic sample to be separated and asecond separation unit downstream of the first separation unit andsupplyable with the fluidic sample after treatment by the firstseparation unit, wherein the control device is configured forcontrolling the first separation unit to execute a primary separationsequence within a measurement time interval for separating the fluidicsample into a plurality of fractions, controlling the second separationunit to execute a plurality of secondary separation sequences within themeasurement time interval for further separating at least a part of theseparated plurality of fractions into a plurality of sub-fractions,wherein the secondary separation sequences form part of a common sampleseparation method defined by a common specification of the sampleseparation involving a set of parameters, and adjusting, over a progressof the primary separation sequence, at least one parameter according towhich at least one of the plurality of secondary separation sequences isexecuted.

According to another exemplary embodiment of the present invention, asample separation apparatus for separating a fluidic sample is provided,wherein the sample separation apparatus comprises a first separationunit supplyable with the fluidic sample to be separated, a secondseparation unit downstream of the first separation unit and supplyablewith the fluidic sample after treatment by the first separation unit,and a control device having the above mentioned features for controllingoperation of the first separation unit and the second separation unit.

According to still another exemplary embodiment of the presentinvention, a process of separating a fluidic sample by a firstseparation unit supplyable with the fluidic sample to be separated andby a second separation unit downstream of the first separation unit andsupplyable with the fluidic sample after treatment by the firstseparation unit is provided, wherein the process comprises controllingthe first separation unit to execute a primary separation sequencewithin a measurement time interval for separating the fluidic sampleinto a plurality of fractions, controlling the second separation unit toexecute a plurality of secondary separation sequences within themeasurement time interval for further separating at least a part of theseparated plurality of fractions into a plurality of sub-fractions,wherein the secondary separation sequences form part of a common sampleseparation method defined by a common specification of the sampleseparation involving a set of parameters, and adjusting, over a progressof the primary separation sequence, at least one parameter according towhich at least one of the plurality of secondary separation sequences isexecuted.

According to still another exemplary embodiment of the presentinvention, a software program or product is provided, preferably storedon a data carrier, for controlling or executing the process having theabove mentioned features, when run on a data processing system such as acomputer.

Embodiments of the invention can be partly or entirely embodied orsupported by one or more suitable software programs, which can be storedon or otherwise provided by any kind of data carrier, and which might beexecuted in or by any suitable data processing unit. Software programsor routines can be preferably applied in the context of measurementcontrol and measurement data analysis. The measurement control andmeasurement data analysis scheme according to an embodiment of theinvention can be performed or assisted by a computer program, i.e. bysoftware, or by using one or more special electronic optimizationcircuits, i.e. in hardware, or in hybrid form, i.e. by means of softwarecomponents and hardware components.

In the context of this application, the term “fluidic sample” mayparticularly denote any liquid and/or gaseous medium, optionallyincluding also solid particles, which is to be analyzed. Such a fluidicsample may comprise a plurality of fractions of molecules or particleswhich shall be separated, for instance biomolecules such as proteins.Since separation of a fluidic sample into fractions involves a certainseparation criterion (such as mass, size, volume, chemical properties,charge etc.) according to which a separation is carried out, eachseparated fraction may be further separated by another separationcriterion (such as mass, size, volume, chemical properties, chargeetc.), thereby splitting up or separating a separate fraction into aplurality of sub-fractions.

In the context of this application, the term “fraction” may particularlydenote such a group of molecules or particles of a fluidic sample whichhave a certain property (such as mass, volume, chemical properties,etc.) in common according to which the separation has been carried out.However, molecules or particles relating to one fraction can still havesome degree of heterogeneity, sometimes simply because resolution offirst separation process is not sufficient enough, but i.e. can befurther separated in accordance with another separation criterion.

In the context of this application, the term “sub-fractions” mayparticularly denote individual groups of molecules or particles allrelating to a certain fraction which still differ from one anotherregarding a certain property (such as mass, volume, chemical properties,etc.). Hence, applying another separation criterion for the secondseparation as compared to the separation criterion for the firstseparation allows these groups to be further separated from one anotherby applying the other separation criterion, thereby obtaining thefurther separated sub-fractions.

In the context of this application, the term “downstream” mayparticularly denote that a fluidic member located downstream compared toanother fluidic member will only be brought in interaction with afluidic sample after interaction with the other fluidic member (hencebeing arranged upstream). Therefore, the terms “downstream” and“upstream” relate to a flowing direction of the fluidic sample. In termsof the sample separation apparatus, the second separation unit islocated downstream of the first separation unit.

In the context of this application, the term “sample separationapparatus” may particularly denote any apparatus which is capable ofseparating different fractions of a fluidic sample by applying a certainseparation technique. Particularly, two separation units may be providedin such a sample separation apparatus when being configured for atwo-dimensional separation. This means that the sample is firstseparated in accordance with a first separation criterion, and issubsequently separated in accordance with a second, different,separation criterion or a different selectivity of the same criterion(see RP-RP separations with different temperature on the two columns).

The term “separation unit” may particularly denote a fluidic memberthrough which a fluidic sample is transferred and which is configured sothat, upon conducting the fluidic sample through the separation unit,the fluidic sample will be separated into different groups of moleculesor particles (called fractions or sub-fractions, respectively). Anexample for a separation unit is a liquid chromatography column which iscapable of trapping and selectively releasing different fractions of thefluidic sample.

In the context of this application, the term “primary separationsequence” may particularly denote a separation method or a part thereofaccording to which a fluidic sample is to be separated in the firstseparation unit. This may include a plurality of steps to be carried outsubsequently. The execution of these steps occurs over a so-calledmeasurement time interval. In a preferred embodiment, the primaryseparation sequence is a gradient run by which the fluidic sample isseparated in the first separation unit by changing a ratio of two (ormore) solvents gradually, thereby selectively trapping and laterreleasing individual fractions of the fluidic sample on the firstseparation unit.

In the context of this application, the term “plurality of secondseparation sequences” may particularly denote sequences having a similaror the same characteristic as the first sequence but which are to beexecuted by the second separation unit. Furthermore, each of the secondseparation sequences is executed over a time interval being smaller thanthe measurement time interval relating to the primary separationsequence. In other words, several or many secondary separation sequencesmay be carried out within a time interval of the primary separationsequence. This means that the fluidic sample is split or separated intothe various fractions during execution of the primary separationsequence, whereas the secondary separation sequences chop the separatedfractions into further subsections by applying another, at leastpartially different separation criterion. For instance, a number ofsecondary separation sequences relating to one primary separationsequence may be in a range between 5 and 3000, particularly between 10and 500.

In the context of this application, the term “(primary) measurementinterval” may particularly denote a time interval required for executingthe primary separation sequence. Such a time interval may be in a rangebetween 1 min and 5 h, particularly between 5 min and 1 h. It may relateto the time required for executing a gradient run on a first separationunit configured as a liquid chromatography column. In accordance withthe long-lasting primary separation sequence, the sample can beseparated into a plurality of fractions by a first separation criteria(for instance the size). In the subsequent, at least partiallyorthogonal secondary separation sequences, each fraction separatedduring the primary separation sequence can be further separated into aplurality of sub-fractions (particularly in accordance with anotherseparating criterion like chemical property such as solubility).

In the context of this application, the term “progress of the primaryseparation sequence” means the time period between a start and theactual measurement time interval. In parallel there are several secondseparation sequences performed, which hold a parameter that depends onprogress of the primary separation. Therefore, during execution of theprimary separation sequence, an adjustment of the parameters of thesecondary separation sequences, may be performed. This may require orsimply include that the progress (or clock) of the primary separationunit is communicated to the second separation unit, or vice versa.

In the context of this application, the term “parameter according towhich one of the plurality of separation sequences is executed” mayparticularly denote one or more quantitative values (such as a value ofa solvent composition, a temperature, the duration of a secondaryseparation sequence) and/or one or more qualitative measurementparameters (such as an operation mode according to which a separationunit is operated) which can be changed under control of the control unitand/or based on user input over the duration of the measurement timeinterval.

In the context of this application, the term “common sample separationmethod defined by a common specification of the sample separationinvolving a set of parameters” may particularly denote a workflow, analgorithm or a set of operation parameters defining as to how a sampleseparation apparatus is to be operated or run. Thus, the common sampleseparation method may include a complete set of data which, whenprovided to the sample separation apparatus, defines a dedicatedoperation of this sample separation apparatus. For example, the commonsample separation method may define a procedure of separating differentcomponents of fluids by the sample separation apparatus (for example arecipe as to how to run a liquid chromatography, gas chromatography orgel electrophoresis experiment) or a procedure requiring officialapproval (for instance an approval procedure before the FDA, Food andDrug Administration, in the United States), a procedure of flushing thedevice (for example an algorithm according to which a rinse solution issupplied for removing traces of fluids from a previous investigation,thereby suppressing undesired crosstalk or contamination), a selectionof a solvent for the sample separation apparatus (for instance selectingmultiple constituents of such a solvent, their relative concentrations,etc.), a procedure of applying a concentration gradient to the sampleseparation apparatus (for example to perform a liquid chromatographyanalysis using a chromatographic column) and/or a selection of anoperation temperature (and/or other physical parameters such aspressure) for the sample separation apparatus. The operation mode maydefine a sequence of instructions providable to the sample separationapparatus for operating the sample separation apparatus. Such a set ofinstructions may be sufficient for running the fluidic device inaccordance with a desired scheme. Particularly in the field oftwo-dimensional liquid chromatography, the sample separation may be achromatographic method.

According to an exemplary embodiment of the invention, a two-dimensionalseparation of a fluidic sample, i.e. a separation of a fluidic samplebased on two different separation criteria, is carried out in aparameterizable manner, what concerns the separation in the seconddimension. Normally a gradient is programmed as a timetable (TTBL) inwhich at specific time points fixed values are commanded, but now in asimple approach e.g. in this TTBL so-called ‘key’-values are entered,for which at the given execution time the progress of the firstdimension separation is monitored, from which these key-values arefilled in. The progression or gradient of these key-values may be givenin a TTBL. This gives a user-friendly and intuitive way of defining suchprogressive execution protocol. In other words, not a strict executionof certain primary and secondary separation sequences is required, butin contrast to this the second dimension of such a two-dimensionalseparation system is adjusted, for instance continuously, bymanipulating a parameter set. For example, the result of a correspondingtwo-dimensional separation result is plottable along a two-dimensionalcoordinate system, plotting a first separation criterion (such as aretention time of a first liquid chromatography separation) along oneaxis and plotting a second separation criterion (for instance aretention time according to a second, different liquid chromatographyseparation) along the second axis. An equal distribution of theindividual peaks relating to individual fractions and sub-fractions overthis two-dimensional area assumes that two separation criteria arecompletely orthogonal, i.e. that there is no correlation between them.This is however not always true in reality. For instance, one may thinkabout a first separation criterion based on mass and a second separationcriterion based on volume of the particles. It is quite unlikely thatthe particles with an extraordinarily high mass have at the same time anextraordinarily small volume. In view of such partial deviations fromcomplete orthogonality between the two separation criteria, thearrangement of the peaks over the two-dimensional plotting area is nothomogeneous, thereby not using efficiently the two-dimensional plot.Furthermore, value of the measurement time is lost in specific regionsof a two-dimensional plot (relating to the measurement) in areas inwhich only few or no peaks of interest can be found. Exemplaryembodiments of the invention now address this phenomenon and allow theparameters of the secondary separation sequence to be adjusted for moreefficiently using resources in terms of measurement time, sampleseparation apparatus and display of results thereof. The hierarchicalapproach of defining the course of action of the secondary separationsequences in terms of the progress of the primary separation sequencehas turned out to be capable of rendering the two-dimensional separationprocedure more efficient and flexible for a user.

By configuring the sample separation system so that the secondaryseparation sequences form part of a common sample separation methoddefined by a common specification of the sample separation involving aset of parameters (for instance one data set stored in one file), it maybe ensured that the separation method remains always the same, i.e. isnot changed, over execution of the sample separation or during themeasurement time interval. A skilled person will understand that amethod change will require a significant amount of time (for example atleast 10 seconds) so that changing a method for adjusting a parameterover a progress of the first separation sequence would involvesignificant delay and may even disturb the sample separation or maydeteriorate separation performance. In contrast to this, an embodimentof the invention defines at least all secondary separation sequences interms of a single common separation method simultaneously allowing foran adjustment of parameters of the secondary separation sequences overthe progress of the first separation sequence.

Particularly, such an embodiment can be denoted as a tune-type parameteradjustment concept. While in the above example regarding the key values,the systems works more like a look-up-table, the concept of tune-typeparameters has more of a self-perpetuating character. For instance, sucha concept may define development of a parameter by a rule such as “startat 5, increment by 2% until 8 is reached”. Also a complex mathematicalfunction may be defined here which may even consider data or resultsfrom previous separation experiments or expert rules. It is for examplepossible to perform a scouting run, wherein a present peak distributionis used for improving or optimizing the use of a two-dimensional displayarea. It is possible to program shapes such as gradients in the seconddimension by using placeholders which may assume different values overthe progress, the running time or measurement time interval, of thefirst dimension. Thus, selective adaptation or adjustment of manysecondary separation sequences can be enabled by only a very limited setof points of support (or sampling points), or even by a relation orfunction.

In the following, further exemplary embodiments of the control devicewill be explained. However, these embodiments also apply to the sampleseparation system, the process, and the software program or product.

In an embodiment, the common specification of the common sampleseparation method comprises a parameterized shape relation defined forat least two of the, particularly for all of the, secondary separationsequences in common, and a development instruction defining theparameters of the shape relation for the at least two, particularly forall, of the secondary separation sequences over a progress of the firstseparation sequence The term “parameterized shape relation” (orenvelope) may particularly denote a relation between experimentalvariables (such as measurement time and solvent composition) defining aprocess flow for the group of the two or all secondary separationsequences. For instance in terms of a chromatographic gradient run, eachsecondary separation sequence may be defined by the general shape of aslow rising edge followed by a fast falling edge, possibly with constantsections in between. One or more parameters may individualize such ageneral shape relation differently for different second separationsequences. In other words, the general shape relation may be the samefor the group of two or all secondary separation sequences, whereas itsparameters may be different for different secondary separationsequences, thereby individualizing or adjusting them for an individualsecondary separation sequence. In an embodiment, the shape relation maybe a shape function. At least a part of the parameters of the shaperelation may be at least part of the parameter or parameters which is orare adjusted over the progress of the primary separation sequence.

In an embodiment, the development instruction comprises a developmentrelation defining development of the parameters, i.e. defining anabstract relation between parameter values and a number of a secondaryseparation sequence in a chronological order. The term “developmentrelation” may particularly denote a (mathematic) relation, moreparticularly a (mathematic) function, defining development of theparameters for each secondary separation sequence in terms of a relationor a rule. For instance, the development relation may be a polynomialfunction or a trigonometric function. For adjusting development of acertain parameter over a number of, for instance fifty, secondaryseparation sequences, it may be sufficient for a user to simply definethe development instruction in abstract terms without the need tomanually type in a huge number of parameter values for each secondaryseparation sequence separately (as would be required if each of thesecondary separation sequences would be configured as a separateseparation method). This makes the task of adjusting parameters, or evenre-adjusting parameters of existing methods, feasible for a user. In anembodiment, the development instruction may comprises a sample specificshape and progression calculated based on data from a previousanalytical separation. Thus, knowledge from historical experiments mayalso be used as a basis for determining the development characteristic.

Additionally or alternatively, the development instruction comprises aset of specific parameter values, each being assigned to a specificsecondary separation sequence in a chronological order, stored in adevelopment database. A development database (such as a lookup table)may define the parameters for each secondary separation sequence interms of specific parameter values. Thus, user guidance may besignificantly improved by the concept of depositing the parameters in amachine-readable manner without the need for a user to manually inputindividual parameters for each of, for instance fifty, secondaryseparation sequences. Therefore the concept of development instructionsmay make the adaptation of the secondary separation sequence over themeasurement time interval manageable.

In an embodiment, the primary separation sequence forms part of the samecommon sample separation method as the secondary separation sequences.Hence, the entire two-dimensional separation may be performed in termsof a single common separation method, i.e. one common set ofinstructions and parameters, rendering parameterization feasible andhandling fast. For a liquid chromatography embodiment, this means thatboth the primary as well as the secondary separation sequences may bedefined in terms of a common chromatographic method defining thetwo-dimensional liquid chromatography separation as a whole or in anintegrated architecture.

In an embodiment, the control device is configured for adjusting the atleast one parameter so that gradient runs, as the plurality of secondaryseparation sequences, perform a drift, particularly a continuous drift,while another gradient run is executed as the primary separationsequence. In such an embodiment, a number of gradient curves are carriedout as the secondary separation sequences. Such a gradient (as anexample for a shape function) may start at a base value (as an examplefor an adjustable parameter of the shape function), may continue with acertain solvent composition up to a final value (as an example for anadjustable parameter of the shape function) along a for instance linearcurve, may stay constant for a while (as an example for an adjustableparameter of the shape function) and may then go back down again to theinitial base value of the solvent composition. In the describedembodiment, a number of secondary separation sequences now may usecorrespondingly shaped time dependencies of the solvent compositionwhich may however be shifted relative to one another along a verticalaxis defining solvent composition. Therefore, in a plotting diagram, thefocus of the measurement time may be set on plotting sections in whichmany or particularly interesting species are expected. This allows tomore efficiently utilize resources in terms of measurement time,software and hardware.

In an embodiment, at least one of the primary separation sequence andthe plurality of secondary separation sequences relate to achromatographic gradient run. In such an embodiment, both separationunits are chromatographic separation columns filled with beads fortrapping and selectively releasing individual fractions of the fluidicsample. The two columns may differ, for instance with regard to theirchemical interaction with the fluidic sample, particle size, porosity,temperature or any other separation-related property. However, in otherembodiments, other kinds of separation techniques may be combined, forinstance chromatography with electrophoresis, mass spectroscopy, etc.

In an embodiment, at least one of the plurality of secondary separationsequences is parameterized, wherein at least a part of correspondingparameters is adjusted over the progress of the primary separationsequence in accordance with a predefined progress rule. Such a progressrule may be an abstract rule (such as a relation, a function, aniterative rule, or a decision criterion) in accordance with which avalue of the parameter develops with ongoing primary separation sequenceprogressing over time. Therefore, particularly the parameters indicatingthe individual secondary separation sequences can be adjusted. However,additionally or alternatively, also an adjustment of the primaryseparation sequence can be performed, under control of a user and/or ofthe control unit.

In an embodiment, at least two, particularly each, of the plurality ofsecondary separation sequences relate to a parameterized shape relationdefined as a gradient curve starting from a first local extreme value(as a reference point, such as a local minimum or base value, or a localmaximum or top value), subsequently rising or falling to a second localextreme value (as another reference point, such as a local maximum ortop value, or a local minimum or base value) and then falling or risingto a next first local extreme value (local minimum or local maximum),wherein at least a part of corresponding parameters of the parameterizedgradient curves is adjusted over the progress of the primary separationsequence. More specifically, at least two, particularly each, of theplurality of secondary separation sequences relates to a parameterizedshape relation defined as a gradient curve starting from a base point,subsequently rising to a top point and then dropping to a next basepoint, wherein at least a part of corresponding parameters of theparameterized gradient curves is adjusted over the progress of theprimary separation sequence. Hence, the above-mentioned shape relationmay define a chromatographic gradient run in abstract terms. In such anembodiment, the parameters may be the solvent composition at the basepoint, the solvent composition of the top point, the slope of thegradient, the time interval for advancing from the bottom point to thetop point, etc. For example, the solvent composition of the base pointmay be adjusted across the primary separation progress for every othersecondary separation sequences in accordance with a continuouslyincreasing function. In an alternative embodiment, it is also possiblethat at least two, particularly each, of the plurality of secondaryseparation sequences relates to a parameterized shape relation definedas a gradient curve starting from a top point, subsequently dropping toa base point and then rising to a next top point, wherein at least apart of corresponding parameters of the parameterized gradient curves isadjusted over the progress of the primary separation sequence. Such anembodiment may relate to a scenario in which the gradient runs in theopposite direction as compared to the previous embodiment, for examplefrom 95% to 40% (for instance in the case of hydrophilic interactionliquid chromatography, HILIC). In a further alternative embodiment, thereference points of the secondary separation sequences need not be localextreme values (i.e. local minima or maxima), but can also relate to aposition in the sequence at which a kink (i.e. a position at which thefirst derivative of the gradient curve is not continuous), etc. occursin the gradient curve. Before and after such a kink, the algebraic signof the first derivative of the gradient curve may be the same, whereinthe algebraic sign of the first derivative of the gradient curve changesat a local extreme value.

In an embodiment, the control device is configured so that the firstlocal extreme values (base points and/or the top points) are adjusted todiffer among different ones of the plurality of secondary separationsequences, particularly to continuously increase (or alternativelycontinuously decrease) along a succession of the plurality of secondaryseparation sequences. In other words, the later a certain secondaryseparation sequence within the primary measurement interval, the higherthe base point will be at which the secondary separation sequence startsfor the next gradient run in the second dimension.

In an embodiment, the primary separation sequence relates to aparameterized shape relation defined as a gradient curve starting from afirst local extreme value, subsequently rising or falling to a secondlocal extreme value, and then falling or rising to the first localextreme value. More specifically, also the primary separation sequencerelates to a parameterized gradient curve starting from a base point,subsequently rising to a top point, and then dropping to the base point,or vice versa. Hence, not only the secondary separation sequences may beparameterized, but also the first separation sequence may be defined byone or more parameters which can also be adjusted. Thereby, theflexibility of the system can be further increased.

In an embodiment, the control device comprises a determining unitconfigured for determining a two-dimensional plot representing theseparation of the fluidic sample into the plurality of fractions along afirst dimension and for representing the separation of the separatedplurality of fractions into the plurality of sub-fractions along asecond dimension. In such an embodiment, the two-dimensional plot may bedisplayed on a display device (such as a monitor) for allowing a user tovisually perceive the result of the fluidic sample separation. The twodimensions or axes may be perpendicular so that the separation inaccordance with the first separation unit and therefore first separationcriterion may be plotted for instance along an abscissa, whereas theseparation by a second separation unit may be plotted along an ordinate,or vice versa. Each fraction or sub-fraction may then be visible as acertain peak or spot in this two-dimensional coordinate system.

In an embodiment, the control device is configured for adjusting the atleast one parameter in accordance with a rule or optimization algorithmto increase a degree of homogeneity according to which the sub-fractionsare distributed over the two-dimensional area of the two-dimensionalplot. The adjustment of the one or more parameters may therefore beperformed so as to better use the available two-dimensional display areaof the plots. By skipping or rapidly scanning over regions of thedisplay area in which no peaks, no interesting peaks, or only a verysmall density of peaks is expected or predicted, measurement resourcesmay be used more efficiently.

In an embodiment, the control device is configured for adjusting the atleast one parameter so as to equally distribute the sub-fractions overthe two-dimensional area of the two-dimensional plot. Such an equaldistribution of peaks over a two-dimensional display area may soundmanipulative on a first view. Therefore, in order to indicate to a userthat a rescaling of the axis has been performed as a result of theadjustment of the parameters, a corresponding function according towhich the representation along one of the two axes deviates from alinear representation may be indicated along the axis by markers or witha bar indicating such a density of display by a color code.

In an embodiment, the control device is configured for determininginformation indicative of at least one low-density region of peaksrelating to the sub-fractions over the two-dimensional area of thetwo-dimensional plot, and is configured for adjusting the at least oneparameter so that density of the peaks is increased in the at least onelow-density region (may may be decreased in at least one high-densityregion) of the two-dimensional plot as a result of the adjusting. Bytaking this measure, insufficiently used display area resources may bedetected, and the separation sequence may be adjusted so as to moreefficiently use such regions of displaying.

In an embodiment, the control device is configured for adjusting the atleast one parameter in accordance with a user input. Therefore, it isalso possible to perform the secondary separation sequences inaccordance with user preferences or user selection, for instance in ascenario in which a user is only interested in information regarding acertain portion of the fractions or sub-fractions and is not interestedin other fractions or sub-fractions. Then, the measurement may befocused on such interesting spots which may be then detected moreaccurately or more quickly. Furthermore, also the display characteristicof the individual peaks may be adjusted by a user by correspondinglyadjusting parameters such as slope of a gradient curve, time spent for asecondary separation sequence, specific solvent compositions, etc.

In an embodiment, the control device is configured for adjusting asolvent composition during a chromatographic run as the at least oneparameter. For example, such a solvent may be a mixture of water andacetonitrile (ACN). The solvent composition is then the ratio betweentwo (or more of) such solvents, for instance a ratio between water andACN. The characteristics according to which such a ratio and thereforethe solvent composition changes over time may be parameterized andadjusted over the progress of the primary separation sequence.

In an embodiment, the control device is configured for adjusting a timeto volume characteristic, particularly a flow rate, during achromatographic run as the at least one parameter. For example, the timemay denote the progress of the measurement time interval. The volume orvolume-related parameter may be any fluid volume, for instance of amobile phase and/or the fluidic sample. By adjusting the relationbetween time and volume, the flow rate may be adjusted.

In an embodiment, the control device is configured for adjusting atemperature of the first separation unit and/or the second separationunit as the at least one parameter. Hence, also external parameters suchas temperature or even pressure can be adjusted for the separation unitsso as to modify the separation criteria applied by the two at leastpartially orthogonal separation procedures.

In the following, further exemplary embodiments of the sample separationsystem will be explained. However, these embodiments also apply to thecontrol device, the process, and the software program or product.

In an embodiment, the first separation unit and the second separationunit are configured so as to execute the respective sample separation inaccordance with different separation criteria, particularly inaccordance with at least partially but not completely orthogonalseparation criteria. In this context, the term “orthogonal” mayparticularly denote the conventional but not very accurate understandingthat two different separation criteria in a two-dimensional liquidchromatography system are completely decoupled. This is not the case inpractice, since for instance a separation with regard to mass and aseparation with regard to volume of particles such as molecules are notcompletely independent. Exemplary embodiments of the invention makebenefit of this cognition and propose to adjust the parameters underconsideration of the fact that the separation criteria of the twoseparation units are not completely independent from one another. Thishas an impact on the regions in a two-dimensional chromatogram in whichthe likelihood to derive a high density of fractions is higher than inother regions. This can be addressed by the adjustment of theparameters.

In an embodiment, the first separation unit and the second separationunit are configured so as to execute the respective sample separation onidentical separation media but with different operating conditions. Suchoperating conditions may be different solvents, different steepness ofelution gradients, different column temperatures, different flows and/ordifferent pressures, so that the separation criteria are partially butnot completely orthogonal. However, not or not only the separation unitsmay relate to non-completely orthogonal separation, but additionally oralternatively it is possible that the partial orthogonality is achievedby using a similar or even the same separation technique, but byadjusting the apparatus properties so that a partial orthogonality isobtained. For example, it is possible to use twice the same separationcolumn, but to operate it at different temperature and/or with differentsolvents or solvent compositions.

In an embodiment, the first separation unit and the second separationunit are configured so as to execute the respective sample separation onidentical separation media but with different operating conditions,particularly at least one of the group consisting of different solvents,different steepness of elution gradients, different column temperatures,different flows and different pressures, so that the separation criteriaare partially but not completely orthogonal. However, not or not onlythe separation units may relate to non-completely orthogonal separation,but additionally or alternatively it is possible that the partialorthogonality is achieved by using a similar or even the same separationtechnique, but by adjusting the apparatus properties so that a partialorthogonality is obtained. For example, it is possible to use twice thesame separation column, but to operate it at different temperatureand/or with different solvents or solvent compositions.

In an embodiment, at least one of the first separation unit and thesecond separation unit is configured for performing a separation inaccordance with one of the group consisting of liquid chromatography,supercritical-fluid chromatography, capillary electrochromatography,electrophoresis and gas chromatography. However, other separationtechniques may be applied as well.

In an embodiment, the sample separation apparatus is configured as atwo-dimensional liquid chromatography sample separation apparatus. It ispossible to apply a so-called heart cutting approach. In such anoperation mode, the second dimension (relating to the secondaryseparation sequences) is only carried out specifically and individuallyfor specific regions, for instance since the separation along the firstaxis has shown that it might be worthwhile to further separatesub-fractions of a corresponding fraction. Each of the identifiedfractions might be separated by a different second dimension operatingcondition or sequence.

The first and/or second separation unit may be filled with a separatingmaterial. Such a separating material which may also be denoted as astationary phase may be any material which allows an adjustable degreeof interaction with a sample so as to be capable of separating differentcomponents of such a sample. The separating material may be a liquidchromatography column filling material or packing material comprising atleast one of the group consisting of polystyrene, zeolite,polyvinylalcohol, polytetrafluorethylene, glass, polymeric powder,silicon dioxide, and silica gel, or any of above with chemicallymodified (coated, capped etc) surface. However, any packing material canbe used which has material properties allowing an analyte passingthrough this material to be separated into different components, forinstance due to different kinds of interactions or affinities betweenthe packing material and fractions of the analyte.

At least a part of the first and/or second separation unit may be filledwith a fluid separating material, wherein the fluid separating materialmay comprise beads having a size in the range of essentially 0.1 μm toessentially 50 μm. Thus, these beads may be small particles which may befilled inside the separation section of the microfluidic device. Thebeads may have pores having a size in the range of essentially 0.01 μmto essentially 0.2 μm. The fluidic sample may be passed through thepores, wherein an interaction may occur between the fluidic sample andthe surface of the pores.

The sample separation apparatus may be configured as a fluid separationsystem for separating components of the sample. When a mobile phaseincluding a fluidic sample passes through the fluidic device, forinstance by applying a high pressure, the interaction between a fillingof the column and the fluidic sample may allow for separating differentcomponents of the sample, as performed in a liquid chromatographydevice.

However, the sample separation apparatus may also be configured as afluid purification system for purifying the fluidic sample. By spatiallyseparating different fractions of the fluidic sample, a multi-componentsample may be purified, for instance a protein solution. When a proteinsolution has been prepared in a biochemical lab, it may still comprise aplurality of components. If, for instance, only a single protein of thismulti-component liquid is of interest, the sample may be forced to passthe columns. Due to the different interaction of the different proteinfractions with the filling of the column (for instance using a gelelectrophoresis device or a liquid chromatography device), the differentsamples may be distinguished, and one sample or band of material may beselectively isolated as a purified sample.

The sample separation apparatus may be implemented in differenttechnical environments, like a sensor device, a test device, a devicefor chemical, biological and/or pharmaceutical analysis, a capillaryelectrophoresis device, a capillary electrochromatography device, aliquid chromatography device, a gas chromatography device, an electronicmeasurement device, or a mass spectroscopy device. Particularly, thefluidic device may be a High Performance Liquid device (HPLC) device bywhich different fractions of an analyte may be separated, examinedand/or analyzed.

The sample separation unit element may be a chromatographic column forseparating components of the fluidic sample. Therefore, exemplaryembodiments may be particularly implemented in the context of a liquidchromatography apparatus.

The sample separation apparatus may be configured to conduct the mobilephase through the system with a high pressure, particularly of at least600 bar, more particularly of at least 1200 bar.

The sample separation apparatus may be configured as a microfluidicdevice. The term “microfluidic device” may particularly denote a fluidicdevice as described herein which allows to convey fluid throughmicrochannels having a dimension in the order of magnitude of less than500 μm, particularly less than 200 μm, more particularly less than 100μm or less than 50 μm or less. The sample separation apparatus may alsobe configured as a nanofluidic device. The term “nanofluidic device” mayparticularly denote a fluidic device as described herein which allows toconvey fluid through nanochannels having even smaller dimensions thanthe microchannels.

BRIEF DESCRIPTION OF DRAWINGS

Other objects and many of the attendant advantages of embodiments of thepresent invention will be readily appreciated and become betterunderstood by reference to the following more detailed description ofembodiments in connection with the accompanied drawings. Features thatare substantially or functionally equal or similar will be referred toby the same reference signs.

FIG. 1 illustrates a two-dimensional liquid chromatography systemaccording to an exemplary embodiment.

FIG. 2 illustrates a sample separation apparatus for two-dimensionalliquid chromatography having a control device according to an exemplaryembodiment of the invention.

FIG. 3 shows a diagram indicating separated fractions and sub-fractionsof a fluidic sample as obtained from two-dimensional liquidchromatography.

FIG. 4 shows a diagram indicating a primary separation sequence asperformed by a first liquid chromatography column in a two-dimensionalliquid chromatography apparatus as the one shown in FIG. 2.

FIG. 5 shows a diagram illustrating a plurality of secondary separationsequences as carried out when operating a second chromatographic columnduring the liquid chromatography analysis relating to FIG. 4.

FIG. 6 shows another diagram showing alternative operation of the secondseparation column with other parameters than in FIG. 5.

FIG. 7 shows another example of a primary separation sequence asperformed by an upstream chromatographic column of a two-dimensionalliquid chromatography apparatus.

FIG. 8 shows a corresponding sequence of secondary separation sequencesrelating to the primary separation sequence of FIG. 7 and showingvarious parameters being modifiable so as to adjust the secondaryseparation sequences over the progress of the primary separationsequence according to FIG. 7.

FIG. 9 shows multiple secondary separation sequences superposed toindicate as to how a parameter can be continuously altered over thevarious secondary separation sequences.

FIG. 10 shows a diagram similar to the diagram of FIG. 3 in which theinfluence of the modification of the parameters of the secondaryseparation sequence on the position of peaks and the management of theavailable display area is plotted.

FIG. 11 shows a diagram plotting a primary separation sequence andmultiple assigned secondary separation sequences of a commontwo-dimensional chromatographic method according to an exemplaryembodiment of the invention.

FIG. 12 shows a diagram illustrating a shape function for the multiplesecondary separation sequences according to FIG. 11.

FIG. 13 shows a diagram illustrating a development function definingdevelopment of the parameters of the shape function for the multiplesecondary separation sequences according to FIG. 11 and FIG. 12.

The illustration in the drawing is schematically.

DETAILED DESCRIPTION

In an embodiment of a two-dimensional separation technique, a progressof a first dimension separation is accompanied by a successive orgradual adjustment of parameters along a second dimension. In thefollowing, some basic cognitions of the present inventor will bementioned based on which embodiments of the invention have beendeveloped.

In an embodiment, a two-dimensional liquid chromatography (2D-LC) systemis provided in which a second dimension gradient is adapted to an actualfirst dimension condition. In other words, a second dimension parametermay drift while the first dimension runs a gradient.

In two-dimensional chromatography systems usually the individualseparations are optimized independently. In order to optimize formaximum peak capacity (pc), a preferred goal is to achieve orthogonalseparations in both of the dimensions. Only then the theoreticallyachievable maximum pc value equals the product of both individual pcvalues in the first dimension pc-1^(st) and in the second dimensionpc-2^(nd):

pc=pc-1^(st) *pc-2^(nd)

But very often this is not achieved in practice. Limitations in eitherthe stationary phase available or the character of sample componentscommonly lead to the fact that some common component is found in bothindividual separations. In many examples one may notice that there is asubstantial commonality. Peaks are assorted not randomly across thewhole display space, but more arranged closely around a central line. Inmany cases, there is less chance for a peak elution early in firstdimension and still being very late in second dimension. Likewise peakseluting late in first dimension have low probability to elute early insecond dimension. Consequently, with the very independent operation ofthe two individual separation dimensions one may lose tuning power.

Starting with the second dimension first, in order to fulfill theNyquist sampling criteria it may be desired to run as fast as possible.So it is possible to employ very short analysis cycles: 1. In order tohave decent stacking on the head of the second dimension column it maybe desired to start low with the gradient. 2. In order to ensure thatthe second dimension column is cleaned before the next cycle, one mayraise elution strength to the maximum. A natural result is that one willrun the fast dimension in full span, for RP (reversed phase) separationsusually 0%-100% organic at maximum speed.

According to an exemplary embodiment of the invention, a tune-typecontrol of a sample separation system allows to combine the methods fromboth dimensions. In a situation that there is substantialnon-orthogonality given, this dependent operation may gain peak capacityby the fact that empty spaces in the 2D plot can be used and filled withpeaks. Basically it is possible for a user to zoom into the area wherethere are actually peaks of interest, spreading these given peaks acrossthe field given. A gist is that the method for the second dimension maybe set up in a way, which supports tune-type parameters. Instead of alist of parameters this special second method now contains controlledvariables (in other sections of this application also discussed in termsof parameters and parameterization).

In the following, two cases are discussed:

Case 1): Such variable could be, as a simple example, the columntemperature of the second dimension column. For instance, a user isdoing RP×RP separations with different columns in both dimension. Thereis a certain chance that one column allows separation of substances,which are not separated on the other, and vice versa. In such a regimeit is possible that along the first dimension time, the preselectedsubstances modulated into the second dimension then elute preferentiallypretty late in the second dimension gradient. If a user now programs thecolumn temperature in the second dimension column to raise graduallyfrom one to the other second dimension run, then it is possible tomobilize later sample groups to elute earlier. Some of them may stillelute later, even at high temperatures. So it is advantageously possibleto gain peak capacity.

Case 2): One could have a drifting gradient. Assuming a case in which auser is doing RP×RP separations with different columns in bothdimension, but now the user commands a drift on the gradient settings.Because early in the first dimension time the modulated peak groups showless retention (otherwise they would not appear early), a user may notneed to run all the way to 100% organic to clear the second dimensioncolumn. So a user will command the second dimension gradient slopinginitially 0-60% organic, while with sequential second dimension runs youincrease the top end of composition, gradually from 60% to 100%. up to afinal stage with a gradient 0-100%.

This way it is possible to gain by a more flat gradient elution,separating low retentive peak groups better (filling for instance anupper left region of a display area), while at the same time still onecan employ stronger elution towards the end of the first dimension run.

Likewise it is possible to gain by drifting the initial value of thesecond dimension gradient. While a user may start low in organic toachieve decent stacking for peaks showing low retention, eluting in amatrix of say 10% B, there is a good chance to achieve sufficientstacking still in later first dimension runtimes, where peaks elute in50% B matrix. So a user may command the second dimension gradientsloping initially 0-100% organic, while with sequential second dimensionruns a user may increase the low end of composition, gradually up to40-100%. This way a user may gain by a more flat gradient elution,separating low retentive peak groups better, this time achieving fastersecond dimension elution (filling for instance a lower right region of adisplay area) more towards the end of the first dimension run.

Referring now in greater detail to the drawings, FIG. 1 depicts ageneral schematic of a liquid separation system 10. A first pump 20receives a mobile phase (also denoted as fluid) from a first solventsupply 25, typically via a first degasser 27, which degases and thusreduces the amount of dissolved gases in the mobile phase. The firstpump 20—as a mobile phase drive—drives the mobile phase through a firstseparating device 30 (such as a chromatographic column) comprising astationary phase. A sampling unit 40 can be provided between the firstpump 20 and the first separating device 30 in order to subject or add(often referred to as sample introduction) a sample fluid (also denotedas fluidic sample) into the mobile phase. The stationary phase of thefirst separating device 30 is configured for separating compounds of thesample liquid.

A second pump 20′ receives another mobile phase (also denoted as fluid)from a second solvent supply 25′, typically via a second degasser 27′,which degases and thus reduces the amount of dissolved gases in theother mobile phase. By a fluidic valve 90, the first dimension(reference numerals 20, 30, . . . ) of the two-dimensional liquidchromatography system 10 of FIG. 1 may be fluidically coupled to thesecond dimension (reference numerals 20′, 30′, . . . ). The fluidicsample is separated into multiple fractions by the first dimension, andeach fraction, or a part/slice of it, is modulated into the secondseparation path and further separated into multiple sub-fractions by thesecond dimension.

A detector 50 is provided for detecting separated compounds of thesample fluid. An optional further detector 55 is arranged upstream ofthe valve 90 and may be used for operating the device 10 in aheart-cutting operation. A fractionating unit 60 can be provided foroutputting separated compounds of sample fluid.

While each of the mobile phases can be comprised of one solvent only, itmay also be mixed from plural solvents. Such mixing might be a lowpressure mixing and provided upstream of the pumps 20, 20, so that therespective pump 20, 20′ already receives and pumps the mixed solvents asthe mobile phase. Alternatively, the pump 20, 20′ might be comprised ofplural individual pumping units, with plural of the pumping units eachreceiving and pumping a different solvent or mixture, so that the mixingof the mobile phase (as received by the respective separating device 30,30′) occurs at high pressure and downstream of the pump 20, 20′ (or aspart thereof). The composition (mixture) of the mobile phase may be keptconstant over time, the so called isocratic mode, or varied over time,the so called gradient mode.

A data processing unit 70, which can be a conventional PC orworkstation, might be coupled (as indicated by the dotted arrows) to oneor more of the devices in the liquid separation system 10 in order toreceive information and/or control operation. For example, the dataprocessing unit 70 might control operation of the pump 20, 20′ (e.g.setting control parameters) and receive therefrom information regardingthe actual working conditions (such as output pressure, flow rate, etc.at an outlet of the pump). The data processing unit 70 might alsocontrol operation of the solvent supply 25, 25 (e.g. setting thesolvent's or solvent mixture to be supplied) and/or the degasser 27, 27′(e.g. setting control parameters such as vacuum level) and might receivetherefrom information regarding the actual working conditions (such assolvent composition supplied over time, flow rate, vacuum level, etc.).The data processing unit 70 might further control operation of thesampling unit 40 (e.g. controlling sample injection or synchronizationsample injection with operating conditions of the pump 20). Therespective separating device 30, 30′ might also be controlled by thedata processing unit 70 (e.g. selecting a specific flow path or column,setting operation temperature, etc.), and send—in return—information(e.g. operating conditions) to the data processing unit 70. Accordingly,the detector 50 might be controlled by the data processing unit 70 (e.g.with respect to spectral or wavelength settings, setting time constants,start/stop data acquisition), and send information (e.g. about thedetected sample compounds) to the data processing unit 70. The dataprocessing unit 70 might also control operation of the fractionatingunit 60 (e.g. in conjunction with data received from the detector 50)and provides data back. The data processing unit 70 may include astorage device 75, which allows to store all or selected information ofthe analytical process and also to retrieve stored information (whichmay be advantageous for the above-mentioned scouting operation) fromprevious analytical processes.

In the following, referring to FIG. 2, a two-dimensional liquidchromatography separation system 200 according to an exemplaryembodiment of the invention will be explained in more detail.

Before describing the operation or control modes according to anembodiment of the invention, the structure of the two-dimensional liquidchromatography apparatus 200 will be described. A control unit 202 isprovided which can be a processor (such as a microprocessor or a centralprocessing unit, CPU) or a set of processors and which is configured forcontrolling entire operation of the two-dimensional liquidchromatography apparatus 200. In a hierarchical approach some or all ofthe individual functions may have their own processor, so thatprocessors may then communicate to perform a concerted function (forinstance to enable a second dimension pump to obtain informationregarding the progress of the first dimension). As will be describedbelow in more detail, the control unit 202 centrally controls all othercomponents of the two-dimensional liquid chromatography apparatus 200.This is indicated schematically in FIG. 2 by dotted lines.

A first dimension pump 204 is provided which is capable of pumping adesired solvent composition for a liquid chromatography separationthrough the various fluidic members shown in FIG. 2. In an autosampler206, a fluidic sample to be separated may be injected into the mobilephase provided by the first dimension pump 204. A first dimensionseparation column 208 is arranged downstream of the autosampler 206. Thefirst dimension separation column 208 corresponds to the firstseparation unit 30 shown in FIG. 1. A gradient run, or any otheroperation mode such as an isocratic mode, can be applied to separate thefluidic sample into different fractions which will then be subjected tofurther separation downstream of the first dimension column 208. Fromhere, the separated fractions can be conducted into a first fluidicvalve 210 having different fluidic ports shown as dots in FIG. 2.Furthermore, a number of arcuate grooves are shown at the first fluidicvalve 210 in FIG. 2 interconnecting fluidically with specific ones ofthe ports, depending on the switching state of the first fluidic valve210. The first fluidic valve 210 is formed of two valve members, a rotorand a stator, one of which having the ports and the other having thegrooves. Alternatively it can be formed with two rotors, each having anindependent motion drive. Alternatively the valve function can beachieved by a combination of connected on/off valves. In an operationmode shown in FIG. 2, the separated fluidic sample fraction downstreamthe first dimension column 208 is subsequently guided through the firstfluidic valve 210 into a first loop 218 (which may for instance have avolume of 20 μl). When passing the loop 218, the separated fluidicsample pushes the loop's content through a second fluidic valve 216which is constituted similar to the first fluidic valve 210. From here,the original content of the first loop 218 can be guided into a wastecontainer 220. For comprehensive 2D-LC operation it is desired not tolose any original sample substance to waste. This way quantification ofsample compounds is allowed to be correct.

However, upon correspondingly switching the valves 210, 216, the fluidicsample, after being separated by the first separation column 208 into aplurality of fractions, can be guided through a second loop 214 (havingfor instance an internal volume of 20 μl) while at the same time thesecond dimension pump 212 drives the actual content (slice of a previousfraction) of first loop 218 from there through the second fluidic valve216 into a second dimension separation column 222. In the shownembodiment, as indicated by the dotted box, the second dimension column222 may be arranged in an oven so as to be heated to a temperature offor instance 70° C. during the second dimension separation. Whilepassing the second dimension column 222, each fraction of the fluidicsample which has already been separated by the first dimension column208 may be further separated into a plurality of sub-fractions which canthen be passed to a detector 224 to be detected and/or quantifiedindividually.

In the following, control of the sample separation apparatus 200 will beexplained in further detail.

The apparatus 200 comprises a user interface 250 bidirectionally coupledto the control unit 202 and adapted for allowing a user to select acertain chromatographic method to be executed and for displaying themethod or operation mode as well as measurement results visually on adisplay (such as a liquid crystal display, a cathode ray tube, a plasmadisplay or the like). Such a user interface 250 may include an inputunit such as a touch-sensitive screen, a joystick, a keypad, a button,etc., allowing a user to input commands, parameters, data andinstructions to the control unit 202. With such a user interface 250, auser may design, store and document a way of operating apparatus 200 ina convenient manner.

The sample separation system 200 is configured so that the primaryseparation sequence executed on the first dimension column 208 and thesecondary separation sequences executed on the second dimension column222 form part of a closed, common single chromatographic method definedby a closed, single common specification (or description) of the sampleseparation involving a set of parameters. In other words, thearchitecture of the applied chromatographic method is such that thefirst and the second dimension separation is integral and notsplittable. Hence, during the entire separation procedure or during themeasurement time interval, it can be prevented that the separationmethod needs to be changed by a user during execution of the sampleseparation or during the measurement time interval, which would becumbersome for a user.

This chromatography method, in terms of the second separation sequences,is characterized by a parameterized shape function (see for instanceFIG. 12) defined uniformly for at least two or all secondary separationsequences, i.e. defining a general shape of a time dependence of thesolvent composition as repeated multiple times for separating fractionsinto sub-fractions. One or multiple shape functions among which a usermay make a choice may be stored in a shape function database 252 towhich the control unit 202 has access. The shape function isparameterized which means that only its general shape is predetermined,whereas a number of parameters may be selected so as to individualizethe shape function for each second separation sequence separately. Theseparameters may be described in terms of and derived from a developmentinstruction defining the parameters of the shape function over aprogress of the first separation sequence. Such a developmentinstruction may be defined by means of a development function (see forinstance FIG. 13) stored in a development function database 254 definingdevelopment of the parameters. It is also possible that such adevelopment instruction is defined by means of concrete parameter valuesstored in a development database 256 (defining for each of the secondarysequences an individual parameter set individualizing or defining therespective secondary separation sequence). By this approach of enablingthe control unit 202 to access pre-populated or user-defined databases252, 254, 256 for adapting the secondary separation sequences over aprogress of the primary separation sequence, adaptation of the seconddimension of a 2D chromatographic system is rendered very user-friendlymaking it dispensable for a user to manually choose tens, hundreds orthousands of parameters.

FIG. 3 now shows a two-dimensional chromatogram which can be obtainedwith an arrangement like the one shown in FIG. 2. Along an abscissa 302,a first retention time is plotted, i.e. indicating the separationperformance of the first dimension column 208. Thus, the differentfractions are arranged along the abscissa 302 in diagram 300. Along anordinate 304 of the diagram 300, the sub-fractions each relating to acertain fraction, and as further separated by the second dimensioncolumn 222 are plotted.

As can be taken from FIG. 3, there is accumulation of correspondingpeaks 308 (each of which relates to a certain sub-fraction) close to asymmetry axis 306, i.e. in a central portion of the two-dimensionalplotting area of the diagram 300. In contrast to this, in regions 310and 312, there are no or only few peaks visible. Consequently, themeasurement time spent for regions 310 and 312 is not utilizedefficiently. The reason behind this is that the two separation criteriaapplied by the first dimension column 208 and by the second dimensioncolumn 222 are different, but are not completely independent from oneanother. It is possible to say, both separation criteria share a commoncomponent or a vector. Based on this cognition, the control unit 302 maybe configured in such a manner that the separation procedureparticularly of the second dimension column 222 is adjusted so that theplotting area in FIG. 3 can be used more efficiently, i.e. that eitherthe measurement time and therefore the resources required for measuringareas 310, 312 is utilized and/or that this measurement area is used forplotting interesting parts of the diagram 300 with better selectivityand/or higher accuracy, i.e. with a larger distance between differentpeaks and hence with a better resolution.

For this purpose, the control device 202 controls the first dimensioncolumn 208 to execute a primary separation sequence within a measurementtime interval 420 (which corresponds to the about 30 minutes shown alongthe abscissa 302 in FIG. 3) for separating the fluidic sample into aplurality of fractions.

A corresponding diagram 400 being indicative of the process flow of theprimary separation sequence is shown in FIG. 4. Diagram 400 has anabscissa 402 along which the time is plotted. Along an ordinate 404, asolvent composition is plotted which indicates the time dependence of asolvent composition for a so-called chromatographic gradient run. Thus,starting from a base point 408, the solvent composition is continuouslyincreased along a linear gradient curve 410 until a top point 412 isreached. Then the primary separation sequence is finished and goes backby an almost vertical line 414 to a base point 416, therebyreconditioning the separation column to be prepared for starting a newrun.

Since the liquid chromatography apparatus 200 is configured fortwo-dimensional chromatography, the control device 202 also controls theoperation of the second dimension column 222 in a more rapid way so thata plurality of secondary separation sequences is carried out during themeasurement time spent for the primary separation sequence illustratedin FIG. 4. This is shown in more detail for instance in FIG. 5 whichillustrates a diagram 500 indicating the various secondary separationsequences 504, 506, 508, as executed on the second dimension column 222under control of the control device 202. Along an abscissa 502, the timeis plotted. Along an ordinate 404, the time dependence of the solventcomposition during the multiple gradient runs executed as the secondaryseparation sequences is plotted as well. As can be taken from FIG. 5,during the measurement time of the primary separation sequence 400 shownin FIG. 4, multiple secondary separation sequences 504, 506, 508 areexecuted. By each of these secondary separation sequences 504, 506, 508one of the fractions separated by the first dimension column 208, slicedby the modulator valve 90, is further separated into the plurality ofthe sub-fractions by second dimension column 222.

In the embodiment shown in FIG. 5, the base points of the variousgradient runs 504, 506, 508, . . . continuously increase, as indicatedby reference numeral 510.

Coming back to FIG. 3 and the measurement in areas 310, 312, suchmeasurement regions can be avoided by allowing the control device 202,in a self-acting manner or under control of a user, to modify theparameters according to which the various secondary separation sequences(504, 506, 508 in FIG. 5) operate. Although not shown with referencenumerals in FIG. 5, also these gradient runs can be identified byparameters such as the ones shown for the primary separation sequence ofFIG. 4 with reference numerals 408, 410, 412, 414, 416.

FIG. 6 shows an alternative diagram 600 which is similar to diagram 500with the exception that the various secondary separation sequences 602,604, 606, . . . are now manipulated with regard to their parameters ofthe respective extensions along the abscissa 502. This will have anotherimpact on the distribution of the peaks in diagram 300.

FIG. 7 shows a diagram 700 indicating another primary separationsequence but being very similar to FIG. 4. In correspondence with thisprimary separation sequence, the various parameters which can bemodified in the corresponding secondary separation sequences are shownin a diagram 800 of FIG. 8 having an ordinate 802. Particularly, theindicated parameters x, y, z can be adjusted for each of the secondaryseparation sequences individually.

FIG. 9 shows the story in a different viewing angle. The abscissa now isthe second dimension time, with sequential cycles overlaid. The arrow902 indicates the progress of the first dimension separation. Here isdepicted an advantageous embodiment in which the base points of thevarious gradient runs of the secondary separation sequences arecontinuously increased over the progress of the measurement timeinterval and of the primary separation sequence. This is indicatedschematically by an arrow 902 showing the development of the parametersof the sequential secondary separation sequences (1), (2), (3), (4), and(5).

An impact of an appropriate parameterization and parameter selection forsecondary separation sequences along a progress of a primary separationsequence can be derived from FIG. 10. As indicated by a double arrowhere, individual peaks 308 may be shifted upon correspondingly varyingthe parameters of the secondary separation sequence.

FIG. 11 shows a diagram 1100 overlaying a primary separation sequence1102 and multiple secondary separation sequences 1104 according to anexemplary embodiment of the invention. Along an abscissa 1106, a time isplotted. Along an ordinate 1108 of the diagram 1100, a solventcomposition (ACN) is plotted. A first time section 1120, a second timesection 1130, a third time section 1140, and a fourth time section 1150are distinguished. A parameter defining a base point or start point forthe gradient runs of the secondary separation sequences 1104 iscontinuously increased along a progress of the primary separationsequence 1102.

FIG. 12 shows a diagram 1200 illustrating a shape function 1202 for themultiple secondary separation sequences 1102 according to FIG. 11. Alongan abscissa 1204, a time is plotted. Along an ordinate 1206 of thediagram 1200, a solvent composition (ACN) is plotted. The parameterizedshape function 1202 is defined by parameters P1, P2, P3.

FIG. 13 shows a diagram 1300 illustrating a development function 1302defining development of parameters P1 to P3 of the shape function 1202for the multiple secondary separation sequences 1104 according to FIG.11 and FIG. 12. For each of the sections 1120, 1130, 1140, 1150, therespective development of the parameters P1 to P3 is plotted. ParametersP1 to P3 remain constant in section 1120, slightly increase in section1130, strongly increase in section 1140, and again slightly increase insection 1150. Alternatively, it is also possible that individual shapefunctions describe development of parameters P1 to P3 individually, bothin terms of their magnitude and in terms of the timing relation (asshown e.g. FIG. 5 and FIG. 6).

It should be noted that the progression of the multiple secondaryseparation sequences along the first dimension separation time may bedefined also by complex mathematical terms or functions, e.g. shapecould be given as a series of Bezier functions and the progressivedevelopment of their parameters could be defined by a trigonomicalfunction.

Alternatively the control unit 202 may use the results from a previousanalytical run to construct an optimal pattern of sequential secondaryseparation sequences with the target to utilize the separation spacemore effectively.

It should be noted that the term “comprising” does not exclude otherelements or features and the “a” or “an” does not exclude a plurality.Also elements described in association with different embodiments may becombined. It should also be noted that reference signs in the claimsshall not be construed as limiting the scope of the claims.

1. A control device for a sample separation apparatus for separating afluidic sample, the sample separation apparatus comprising a firstseparation unit supplyable with the fluidic sample to be separated and asecond separation unit downstream of the first separation unit andsupplyable with the fluidic sample after treatment by the firstseparation unit, wherein the control device is configured for:controlling the first separation unit to execute a primary separationsequence within a measurement time interval for separating the fluidicsample into a plurality of fractions; controlling the second separationunit to execute a plurality of secondary separation sequences within themeasurement time interval for further separating at least a part of theseparated plurality of fractions into a plurality of sub-fractions,wherein the secondary separation sequences form part of a common sampleseparation method defined by a common specification of the sampleseparation involving a set of parameters; adjusting, over a progress ofthe primary separation sequence, at least one parameter according towhich at least one of the plurality of secondary separation sequences isexecuted.
 2. The control device according to claim 1, wherein the commonspecification of the common sample separation method comprises: aparameterized shape relation defined uniformly for at least two of the,particularly for all of the, secondary separation sequences; and adevelopment instruction defining the parameters of the shape relationfor the at least two, particularly for all, of the secondary separationsequences over a progress of the first separation sequence.
 3. Thecontrol device according to claim 2, wherein the development instructioncomprises one of the group consisting of: a development relation,particularly a development function, defining development theparameters; and progressive parameter values for the parameters storedin a development database; and a sample specific shape and progressioncalculated based on data from a previous analytical separation.
 4. Thecontrol device according to claim 1, wherein the primary separationsequence forms part of the same common sample separation method as thesecondary separation sequences.
 5. The control device according to claim1, wherein the control device is configured for adjusting the at leastone parameter so that gradient runs, as the plurality of secondaryseparation sequences, perform a drift, particularly a continuous drift,while another gradient run is executed as the primary separationsequence.
 6. The control device according to claim 1, wherein at leastone of the primary separation sequence and the plurality of secondaryseparation sequences relates to a chromatographic gradient run.
 7. Thecontrol device according to claim 1, wherein at least one of theplurality of secondary separation sequences is parameterized, wherein atleast a part of corresponding parameters is adjusted over the progressof the primary separation sequence in accordance with a predefinedprogression rule.
 8. The control device according to claim 1, wherein atleast two, particularly each, of the plurality of secondary separationsequences relate to a parameterized shape relation defined as a gradientcurve starting from a first local extreme value, subsequently rising orfalling to a second local extreme value and then falling or rising to anext first local extreme value, wherein at least a part of correspondingparameters of the parameterized gradient curves is adjusted over theprogress of the primary separation sequence.
 9. The control deviceaccording to claim 8, wherein the control device is configured so thatthe first local extreme values are adjusted to differ among differentones of the plurality of secondary separation sequences, particularly tocontinuously increase or to continuously decrease along a succession ofthe plurality of secondary separation sequences.
 10. The control deviceaccording to claim 8, wherein the primary separation sequence relates toa parameterized shape relation defined as a gradient curve starting froma first local extreme value, subsequently rising or falling to a secondlocal extreme value, and then falling or rising to the first localextreme value.
 11. The control device according to claim 1, comprising adetermining unit configured for determining a two-dimensional plotrepresenting the separation of the fluidic sample into the plurality offractions along a first dimension and for representing the separation ofthe separated plurality of fractions into the plurality of sub-fractionsalong a second dimension.
 12. The control device according to claim 11,wherein the control device is configured for adjusting the at least oneparameter so as to increase a degree of homogeneity according to whichthe sub-fractions are distributed over the two-dimensional area of thetwo-dimensional plot.
 13. The control device according to claim 11,wherein the control device is configured for adjusting the at least oneparameter so as to equally distribute the sub-fractions over thetwo-dimensional area of the two-dimensional plot.
 14. The control deviceaccording to claim 11, wherein the control device is configured fordetermining information indicative of at least one local low-densityregion of peaks relating to the sub-fractions over the two-dimensionalarea of the two-dimensional plot, and is configured for adjusting the atleast one parameter so that density of the peaks is increased in the atleast one local low-density region of the two-dimensional plot as aresult of the adjusting.
 15. The control device according to claim 1,wherein the control device is configured for adjusting the at least oneparameter in accordance with a user input.
 16. The control deviceaccording to claim 1, comprising at least one of the following features:the control device is configured for adjusting a solvent compositionduring a chromatographic run as the at least one parameter; the controldevice is configured for adjusting a time to volume characteristic,particularly a flow rate, during a chromatographic run as the at leastone parameter; the control device is configured for adjusting atemperature of at least one of the group consisting of the firstseparation unit and the second separation unit as the at least oneparameter.
 17. A sample separation apparatus for separating a fluidicsample, the sample separation apparatus comprising a first separationunit supplyable with the fluidic sample to be separated; a secondseparation unit downstream of the first separation unit and supplyablewith the fluidic sample after treatment by the first separation unit; acontrol device according to claim 1 for controlling the first separationunit and the second separation unit.
 18. The sample separation apparatusaccording to claim 17, comprising at least one of the followingfeatures: the first separation unit and the second separation unit areconfigured so as to execute the respective sample separation inaccordance with different separation criteria, particularly inaccordance with at least partially but not completely orthogonalseparation criteria; the first separation unit and the second separationunit are configured so as to execute the respective sample separation onidentical separation media but with different operating conditions,particularly at least one of the group consisting of different solvents,different steepness of elution gradients, different column temperatures,and different pressures, so that the separation criteria are partiallybut not completely orthogonal; at least one of the first separation unitand the second separation unit is configured for performing a separationin accordance with one of the group consisting of liquid chromatography,supercritical-fluid chromatography, capillary electrochromatography,electrophoresis and gas chromatography; the sample separation apparatusis configured as a two-dimensional liquid chromatography sampleseparation apparatus; the sample separation apparatus is configured toanalyze at least one physical, chemical and/or biological parameter ofat least one compound of the fluidic sample; the sample separationapparatus comprises at least one of the group consisting of achromatography device, a liquid chromatography device, an HPLC device, agas chromatography device, a capillary electrochromatography device, anelectrophoresis device, a capillary electrophoresis device, a gelelectrophoresis device, and a mass spectroscopy device; the sampleseparation apparatus is configured to conduct the fluidic sample with ahigh pressure; the sample separation apparatus is configured to conductthe fluidic sample with a pressure of at least 100 bar, particularly ofat least 500 bar, more particularly of at least 1000 bar; the sampleseparation apparatus is configured to conduct a liquid fluid; the sampleseparation apparatus is configured as a microfluidic device; the sampleseparation apparatus is configured as a nanofluidic device; at least oneof the group consisting of the first separation unit and the secondseparation unit is configured for retaining a part of components of thefluidic sample and for allowing other components of the fluidic sampleto pass; at least one of the group consisting of the first separationunit and the second separation unit comprises a separation column; atleast one of the group consisting of the first separation unit and thesecond separation unit comprises a chromatographic column; at least apart of at least one of the group consisting of the first separationunit and the second separation unit is filled with a separatingmaterial; at least a part of at least one of the group consisting of thefirst separation unit and the second separation unit is filled with aseparating material, wherein the separating material comprises beadshaving a size in the range of 1 μm to 50 μm; at least a part of at leastone of the group consisting of the first separation unit and the secondseparation unit is filled with a separating material, wherein theseparating material comprises beads having pores having a size in therange of 0.01 μm to 0.2 μm.
 19. A process of separating a fluidic sampleby a first separation unit supplyable with the fluidic sample to beseparated and by a second separation unit downstream of the firstseparation unit and supplyable with the fluidic sample after treatmentby the first separation unit, wherein the process comprises: controllingthe first separation unit to execute a primary separation sequencewithin a measurement time interval for separating the fluidic sampleinto a plurality of fractions; controlling the second separation unit toexecute a plurality of secondary separation sequences within themeasurement time interval for further separating at least a part of theseparated plurality of fractions into a plurality of sub-fractions,wherein the secondary separation sequences form part of a common sampleseparation method defined by a common specification of the sampleseparation involving a set of parameters; adjusting, over a progress ofthe primary separation sequence, at least one parameter according towhich at least one of the plurality of secondary separation sequences isexecuted.
 20. A software program or product, stored on a data carrier,for executing a process according to claim 19, when run on a dataprocessing system.